Tuberculosis (TB) continues to kill millions of people around the world. New tools to prevent and treat this disease are urgently needed. Iron is an essential metal for all forms of life and most bacterial pathogens including Mycobacterium tuberculosis (Mtb) must import iron from its host to survive. We have discovered a new mycobacterial iron uptake pathway whereby Mtb sequesters iron from human heme sources. The characterization of this pathway will provide novel targets for anti-TB drug design, which is our long-term objective. We propose a putative pathway where heme is sequestered from human hemoglobin by a secreted hemophore and transferred across the membrane by heme transporters. Interestingly, nonpathogenic mycobacterium BCG has an attenuated heme uptake pathway compared to Mtb, which may be, in part, attributed to a single amino acid substitution in the BCG hemophore. We propose a structural and biochemical comparison of the hemophores from Mtb and BCG, and their capabilities to acquire and transfer heme. Specific aims: 1) Structural comparison of Mtb and BCG hemophores in the presence and absence of heme. This will be carried out by X-ray crystallography. 2) To characterize the heme environments in both hemophores. Techniques include circular dichroism, UV/vis and electron paramagnetic resonance spectroscopy, and heme affinity calculations for each hemophore. 3) To investigate the heme acquisition and transfer capabilities of the two hemophores, where heme transfer kinetics between proteins will be observed using stopped-flow spectrophotometry. We will also utilize chromatography methods to observe potential protein-protein interactions between heme donors and acceptors. Relevance to public health. TB, caused by the bacterial pathogen Mycobacterium tuberculosis, kills two million people around the world each year according to the WHO, thus novel undiscovered pathways involved in pathogenicity and persistence are necessary to combat this reemerging disease. Iron is essential for all forms of life, and is acquired by mycobacteria via molecules called siderophores. We have shifted this paradigm, as we have identified a novel iron acquisition pathway whereby iron is obtained via the uptake of human heme, opening up new avenues for anti-TB drug development.